WO2001088176A2

WO2001088176A2 - Measurements of enzymatic activity in a single, individual cell in population

Info

Publication number: WO2001088176A2
Application number: PCT/IL2001/000443
Authority: WO
Inventors: Merav Sunray; Naomi Zurgil; Mordechai Deutsch
Original assignee: Bar-Ilan University
Priority date: 2000-05-18
Filing date: 2001-05-17
Publication date: 2001-11-22
Also published as: EP1287160A4; JP2003533209A; WO2001088176A3; US20030211458A1; AU6056501A; EP1287160A2; IL136232A0

Abstract

A process for measuring enzymatic activity in an identified, isolated, intact, single, viable cell. Each of the viable cells is placed within individual identified locations on a carrier of a cytometer having means to measure enzymatic activity of a single viable cell placed in an identified location. The identified isolated cell is exposed to a substrate of an enzyme to be measured, and the rate of product formed or released following every exposure of the cell to same or different concentrations of the substrate is measured. The isolated cell may be exposed to a sequence of at least two different concentrations of the substrate, and for each exposure the rate of product formed or released is measured. Figure (1) is a model of intracellular conversion of a substrate to a product.

Description

MEASUREMENTS OF ENZYMATIC ACTIVITY IN A SINGLE.

INDIVIDUAL CELL IN POPULATION

Field of the Invention

Enzymes are organic catalysts that cause and direct the numerous chemical

reactions that occur in living organisms. Most of chemical changes that occur in

hving cells are caused and controlled by enzymes. Assessing the enzyme activity

in a particular type of cells is therefore one of the principal approaches to the

study of what goes on in the same individual hving cells.

The present invention provides a new process and methodology for measuring

enzymatic activity in intact individual cells. More specifically, it provides the

capabilities for high precision enzymatic kinetic measurements of individual cells

under repeatable substrate exposure conditions. On-line reagent addition, and

controlling other changes in experimental conditions, can be easily accomplished,

and the dynamic changes in individual given cells is monitored in real-time.

Thus, the process of the invention provides a new valuable tool for assessing

enzymatic reaction kinetics, resulting in determination of activity of an

individual enzyme as well as of a series of different enzymes, in specific intact

cells under defined physiologic conditions. In a preferred embodiment of present invention, the substrate is either passively

or actively enters the cell, once inside, it is processed by the assessed intracellular

enzyme to generate detectable product.

In yet another preferred embodiment, the process of present invention is

applicable for measuring simultaneously the enzymatic activity in many

identified individual cells, within same population.

Since enzymes are ubiquitously involved in cellular function, the monitoring of

their reaction kinetics on the level of a single, individual cell may provide

valuable information. For example, in some human diseases, especially heritable

genetic disorders, there may be a deficiency or even a total absence of one or more

enzymes in the tissue. Moreover, measurements of the cellular activity of certain

enzymes are important in diagnosing diseases. Most enzymes can be poisoned or

inhibited by certain chemical reagents.

Numerous of drugs are designed to inhibit the excessive catalytic activity of

specific enzymes in abnormal conditions. Other drugs inhibit certain enzymes in

malfunctioning cells. The overall activity of such drugs can only be measured in

an intact system of the individual live cell.

An enzymatic activity is usually characterized by two parameters: VMAX - the

maximum enzyme production rate (velocity), of a product (P) out of a substrate (S)

at a saturation concentration of the latter, and KM - the Michaelis - Menten constant, which is reciprocally proportional to the enzyme affinity to the

substrate.

The relation between VMAX , KM, the substrate concentration [S] and the initial

velocity V, at which S converts to P, is given by the Michaelis - Menten equation:

V [s v M. AX κ_M +[sy

Unfortunately Eq. 1 is accurate only for a homogeneous medium in which the

following processes occur: .

[S] + [E] <-» [ES] and [ES] - [P] + [E] where [E] and [ES] are the enzyme

and the complex enzyme - substrate concentrations, correspondingly.

The determination of KM and VMAX, utilizing Eq. 1 calls for sequential exposures

and repeatable measurements of the same individual cell for various values of

[S\.

Unfortunately this requirement can not be achieved by the common cytometers:

The Flow Cytometer (FC) as well as the Laser Scanning Cytometer (LSC). The

FC enables the rapid measurement of the fluorescence intensity ( FI ) of a large

cell population. However because each cell in the flow is measured only once, the

kinetic curves of the FC

1. Dolbcare F, Fluorescent staining of enzymes for flow cytometry, Methods

Cell Biol 33:81-88, 1990

2. Klingel S, Rothe G, Kellerman W, Valet G, Flow cytometric determination

of serine proteinase activities in hving cells with rhodamine 110 substrates, .

Methods Cell Biol 41:449-460, 1994 3. Malin-Berdel J, Valet G, Flow cytometric determination of esterase and phosphatase activities and kinetics in hematopoietic cells with fiuorogenic substrates, Cytometry 1:222-228, 1980

4. Nooter K, Herweijer H, .Jonker RR, van den Engh GJ, On-line flow

cytometry. A versatile method for kinetic measurement, Methods Cell Biol

41:509-526, 1994

5. Turck JJ, Robinson JP, Leucine aminopeptidase activity by flow

cytometry, Methods Cell Biol 41:461-468, 1994

6. Watson JV, Dive C, Enzyme kinetics, Methods Cell Biol 41 :469-508, 1994]

provide sequential measurements of single cells over time but not of the same

single cell. Therefore, investigating different enzyme activities in different cell

types or in subcellular areas using the FC gives only an average KM value for a

population of cells or for specific enzymes in a cell-free system.

The LSC measures the fluorescence kinetic of individual cells under specific

conditions of low cell density in the selected field and of cell types and dyes which

do not suffer from fading, which disrupts the measurement [Watson JV, and Dive

C. Enzyme kinetics. Methods Cell Biol (1994) 41:469-508]. The LSC technique

cannot ensure the accurate rescarming of the same cell after repeatable staining

procedures since the cell may not have preserve its original location. Moreover,

the LSC cannot ensure preservation of the cell locations and thus cell

identification might be lost during repeatable rinsing and exposure to different

substrate concentrations. In order to provide the capabilities for kinetic measurement of individual cells

under repeatable staining conditions, a specially designed cytometer was used.

The cytometer (hereinafter referred to as Cellscan Mark S or CS-S) which, one of

its versions, was described in the US Patents 4,729,949, 5,272,081, 5,310,674 and

5,506,141 found to be applicable for measuring time resolved kinetics of

individual cells during cellular manipulation.

Using the unique apphcation of the CS-S, a new method was developed in which

the same cells are sequentially exposed to increasing substrate concentrations.

The product formation rate is measured for each cell at every substrate

concentration yielding a series of rates for the same individual cell. Using this

data, VMAX and apparent KMAPP (app = apparent) values can be calculated for

each ceU, giving the distribution of KMAPP and VMAX of the measured population

However, it should be emphasized that the process of present invention is not

limited to the CS-S cytometer and any cytometer comprising a microscope, light

detection means, a carrier to which cells are individually located, is within the

scope of the present invention.

Kinetic Analysis:

The kinetic parameters are derived by application of linear and nonlinear

modeling. The linear model y(t)=At+B seeks parameters A and B which fit the

data to a straight line equation, where y(t) is the measured quantity, t is the

time, and A and B are the calculated parameters. The CS-S algorithm uses χ² as

the criteria for goodness-of-fit. a. Single Step Cell Staining;

A simplified model for the description of intracellular turnover of fluorogenic

substrate is presented in Fig. 1. First, the extracellular substrate [ ^L51 ^Jo permeates

into the cell, becoming [S]i - the intracellular substrate concentration. Then [S]i is

hydrolyzed or cleaved by enzymes to yield the intracellular (for example,

fluorescent) product [P]i, which may be released from the cell into the medium

and become [P]o .

As was previously shown [Bedner E, Melamed MR, Darzynkiewicz Z, Enzyme

kinetic reactions and fluorochrome uptake rates measured in individual cells by

laser scanning cytometry, Cytometry 33:1-9, 1998] the kinetics of [P]i can be

described, to a good approximation, by the rate equation :

(2) -^ ^ α - SIo -ZH L

Where α and β are the rates constants for the formation and leakage of the

intracellular fluorescein. It is important to emphasize that α represents two

processes: Permeation of S and its intracellular distribution as well as the

enzymatic hydrolysis of [S]i.

When solving Eq. 2, under the initial condition of one step staining, [P(t=0)]ι = 0

it is easily shown that

(3)

b. Sequential staining:

Another aspect of present invention relates to sequential exposures of the same

individual cells to different substrate concentrations. This differs from the above

case by the fact that at the starting time point of staining, with a given solution,

cells are already being stained to a level of:

τ stands for the time point of terminating the staining with a given substrate

concentration, say M times [S\ (M[S]), and initiation of staining with different

substrate concentration, say N[S\.

Now, it is possible to solve Eq.2 under the initial conditions presented by Eq. 4.

By separation of variables and integration over [P]i between the concentration

limits [P(τ)]i and [P(t)]ι; and integration over time between the time points 0

(when staining solutions are being replaced) and t, one gets:

(a

Converting the logarithmic expression into exponential one and introducing

[F(τ)]ι of Eq.4 into Eq.5 yields:

(6)

F (0] = — M

When single step staining is performed (starting of unstained cell, M=0), only the

last term of Eq. 6 remains, which is consistent with Eq. 3.

As long as the expression exp(-βt) s 1-βt holds for the duration of the observation

interval of the individual cells in given conditions, regardless of their staining

history, each of the exponential terms in Eq. 6 can be replaced, without losing

accuracy, by its first two terms of the power series. Hence, Eq. 6 may be linearly

approximated to give:

(7)

d[F{t

t > τ a[SY (Mτ + Nt) [F(t)]_{T ^ Λ} dt Eq. 7 should be interpreted as follows: for 0< t<τ, staining proceeds according to

[P(t)]i = [S]Mt. After replacing the staining solution M by N at time t=τ, the

staining due to M[S] remain constant [P(τ)]ι = [S]Mτ, While that due to N

increases at a rate of [S N, namely solely depending on the concentration in use.

Simulations of several practical staining protocols, based on Eq.7, are

graphically presented in Fig. 2 and briefly described in the following:

a) Rinsing the cells with a staining solution [N] that maintains [N] = [M, results

in a staining curve [P(t)]ι=α[S]iV[τ+t]. At the observation time τ+t [P]i had a

production rate of α[S]iV, the same rate as that of a[S\M prior to τ+t (Fig. 2a).

b) Rinsing the cells with PBS alone washed away [M\ residues leaving the

staining solution at a concentration [M\ = 0. This action halted any further

production [P]ι (since α[S]iV = 0 at the time of application i) hence [P]i line

remained parallel to the time axis for the duration of the observation t. (Fig. 2b).

c) In a similar way, the cells were rinsed with a staining solution [N\ ≠ [M\ that

washed away [M\ and left the staining solution at a concentration [N]. The

production rate of [P]i, as expected, changed to α N [S] for the observation

duration t. (Fig. 2c )

d) The last experiment, was a combination of b) and c) in succession. First the

cells were rinsed at time ti with PBS and that halted the production of Fi. The

next stage was to rinse with a staining solution [N] ≠ [M\ replacing the PBS with

a solution of concentration [N\. The production rate then changed to α[S]iVfor the

for the observation duration t. (Fig. 2d). Finally, the determination of Δt, the overall sequential staining experiment

procedure time duration, was restricted to follow the present CS-S Standard

deviation in performing individual cell FI measurements, which is < 2%.

In order not to exceed this value when linearly approximating the exponential

terms, a Δt value which keeps the ratio

exp(-βΔt)/(l-βΔt) = 2% is sought. Hence, introducing β = 10-⁴ sec-¹, which is the

outcome of many hundreds of independent experiments (data not shown), yields

Δt ≥ 103 sec.

Summary of the Invention

It is an object of present invention to provide a process for measurement and

characterization of intra- and extra-cellular enzymatic activity taking place in the

same identified individual cell, in a population of cells, following its incubation

with different concentrations of a substrate. The substrate should be a substance

that yields a product that is detectable by physical means, such as changes in

fluorescence intensity, color intensity, radioactive radiation, etc.

It is a further object of present invention to establish a new method for the

determination of KMAPP and VMAX values for enzymatic reactions carried out

inside an identified individual cell. It is an additional object of the present

invention to determine kinetic values of extracellular enzymes, released from an

individual cell. It is yet an additional object of present invention to provide a tool

for measuring differences in kinetic enzymatic activity in the individual cell

following various treatments of same cell with biologically active materials. A further object of present invention is to provide a process for measuring

simultaneously the enzymatic activity in many identified individual cells, within

same population.

Brief Description of the Drawings

Fig. 1: A model of intracellular conversion of a substrate to a product. [S]o, [S]i

are the extracellular and intracellular substrate concentrations and [P]o, [P]ι are

the extracellular and intracellular product concentrations. [E] and [ES] are the

enzyme and enzyme substrate complex concentrations, ki is the rate constant for

formation of the complex [ES], k-i is the rate constant for the reversed reaction

and k₂ is the rate constant for product formation.

Fig. 2: Simulation of an individual cell sequential FI time dependency following

several exposure procedures to substrate concentration. M = multiplication

coefficients of initial substrate concentration.

R = rinsing at a given time point. Panels: a - rinsing with the same concentration

yields identical slopes, b - sequential rinsing with (yield identical slopes as in

panel a) and without (zero slopes) substrate, c - sequential rinsing with

increasing substrate concentrations, d - sequential rinsing with increasing

substrate concentrations while in between rinsing without substrate.

Fig. 3: Experimental results of individual cells sequential staining procedure. The

numbers in the boxes are the slopes of FI(t) (initial velocities), given in arbitrary

intensity units per second. The experiment follows the simulation shown in Fig.

2.

Fig. 4: Complete sequential staining procedure of numerous cells. Each of the four clusters contains 13 lines. Each line defined by six FI measurements taken

in six different time points for the same individual cell when exposed to the

relevant substrate concentration. Ri to R₄ - the space between clusters stands for

replacement duration of the staining solutions (0.6, 1.2, .4 and 3.6.μM). The

solid line in each of the four clusters is sketched for clarification purposes. It

indicates the increasing slopes of one chosen set of sequential exposure of one

individual cell.

Fig. 5: Individual KMAPP and VMAX for two representative cells and their Pearson

correlation coefficient ( ²).

Fig. 6: The distribution of individual KMAPP (6A) and VMAX (6B) for cells that were

incubated with ( — ) and without (- - - ) PHA.

Fig. 7: Rate of change of FI before and after exposure of an individual cell to

hydrogen peroxide (H2O2) compared with control. The ratio pre to post treatment

slopes in control cells is double that of cells exposed to H2O2 (treated).

The following examples are provided merely to illustrate the invention and are

not intended to limit the scope of the invention in any manner.

Examples

Example 1. Measuring intracellular nonspecific esterase activity in a single

lymphocyte using fluorescein-diacetate (FDA) as the substrate. Materials and Methods

Phytohemagglutinin PHA (HA15, Murex Biotech ) was reconstituted in 5 ml of

double-distilled water and further diluted ten times. For stimulation, lOμl of this

solution was added to a 90 μl cell suspension (7xl0⁶ cells/ml).

The culture medium consisted of RPMI-1640 (Biological Industries),

supplemented with 10% (v/v) heat-inactivated fetal calf serum (Biological

Industries), 2mM L-glutamine, 10 mM Hepes buffer solution, ImM sodium

pyruvate, 50 U/ml penicillin and 50Units/ml streptomycin.

A staining solution of 3.6 μM FDA (Riedel-de Haen Ag. Seelze-Hanover) in

Dulbecco Phosphate Buffered Saline (PBS, Biological Industries) was prepared as

follows: 50 mg of FDA was dissolved in 5 ml of DMSO (Sigma). 7.5 μl of this

solution was added to 50 ml PBS. For 0.6, 1.2 and 2.4μM the solution was further

diluted in PBS.

Preparation of Peripheral Blood Mononuclear Cells (PBMC :

30 Heparinized blood (30 ml), was taken from healthy, normal volunteers. The

procedure for separating the PBMC has been described in detail, elsewhere

[Sunray M, Deutsch M, Kaufman M, Tirosh R, Weinreb A, and Rachmani H. Cell

Activation influences cell staining kinetics, Spectrochimica Acta A (1997)

53:1645-1653]. Shortly after removing the iron absorbing cells, the remaining

cells are layered on a two-layer (100% and 80%) cell density gradient (Ficoll

Paque, Pharmacia 1.077 g/ml) and centrifuged. The cells accumulated at the interface between the two Ficoll layers, were collected and kept at 37° C in 5 ml

of enriched culture medium overnight. The next day the PBMC were washed and

resuspended in PBS at a final concentration of 7-10⁶ cells/ml. More than 70% of

the cells were defined as T lymphocytes and viability, which was determined

using eosin, was always higher than 90%.

Activation of PBMC by PHA:

Freshly prepared PBMC (7-10⁶ cells/ml) were incubated at 37°C, 5% C02 with 5

μgr/ml PHA for 30 minutes. PBMC controls were incubated without PHA under

identical conditions.

The CS-S Apparatus

The multiparametric, computerized, discrete cytometer CS-S used in performing

this example was described in detail in the above specified US Patents. Its

central feature is a cell carrier (CC) incorporating a 100 x 100 dimensional array

having a conical cross-section with an upper opening of ~7μm and lower opening

of ~4μm, each approximately 20μm apart, in which individual cells are trapped.

The cell carrier is mounted on a computer-controlled stage that enables repeated

multi-scanning of the same cells.

Cells were irradiated with 1-10 μW of 442 nm light from a He-Cd laser. Under

the staining conditions used here, the scanning time for obtaining a 'count of

10,000 photons in order to have statistical photonic error of ~1% from each,

7 dye-loaded cell varied from 0.001 sec to approximately 0.5 sec. The acquired data, including cell position, measurement duration for each cell,

absolute time, intensity at two different wavelengths, computed fluorescence

polarization values and test set-up information, are displayed on the screen,

on-line, graphically and numerically, and stored in the memory. Software

enables the determination of the range and other statistical characteristics of all

parameters, for either the entire cell population, or an operator-selected

sub -population, or an individual cell, before, or during the scan.

Cell Loading

Loading the cells in wells traps on the Cell Carrier (CC) was carried out, as

described in Deutsch M, and Weinreb A., Apparatus for High Precision Repetitive

Sequential Optical Measurement of Living Cells, Cytometry (1994) 16: 214-226.

An ahquot of 80 μl of unstained cell suspension (7x106 cells/ml) was loaded on the

CC. Initial scanning was then performed in order to detect individual cell

background scattering and auto-fluorescence. This undesired signal is recorded

per measurement location and subtracted from the total emission signal (after

exposure) in order to obtain the correct fluorescence signal.

Cell Staining and Kinetic Measurement:

For fluorescence intensity FI(t) measurements, trapped cells on the CC were

sequentially exposed to increasing concentrations of FDA in PBS staining

solutions.

Following background measurement, the volume of PBS, which covers the cells,

was pumped out and the following procedure was carried out: At time point zero, 40 μl of the lowest substrate concentration solution was

applied on top of the trapped cells and a pre-chosen cell field was sequentially

scanned 6 times. This yielded 6 accurately timed FI data points per each

individual cell at a given dye concentration. FI is usually measured utihzing

epi-fluoesceiice optical arrangement which permits the differentiation between

the excitation energy and the emitted fluorescence energy to be detected by

photomultipliers, CCD detectors etc.

The above procedure is repeated for each different substrate solution used in the

experiment.

This yielded six FI data points for each individual cell, per substrate

concentration, from which V was extracted and the individual cell KMAPP and

VMAX values were calculated. The dead time, i.e., the elapsed time from the

addition of a staining solution to the beginning of the measurement, which is

monitored by the computer, is about 7-15 sec.

Results

Repeatability runs:

The experimental arrangement of the new process calls for high-level

performance in terms of repeatabihty and accuracy in periodical measurements of

individual cells.

The CS-S capability was displayed by performing sequential measurements of FI and FP on 5 min 1.2μM FDA stained trapped cells, following their PBS rinsing

out of excess substrate solution and , possible extra-cellular Pi (at this stage,

constancy of FI is expected due to staining termination and negligibihty of Pi

leakage).

The individual cell coefficient of variance (CV) obtained in more then 10

successive measurement scans of a 10 x 10 cell field, never exceeded 2% for FI.

Fading was not noticeable.

Accuracy Runs:

Accurate intensity measurement capabilities and specific monitoring of

alterations in Fi production rate are mandatory for the present process. This was

first serially examined by measuring FI of the CC loaded with cell-free

fluorescein solutions at concentrations of 0.6, 1.2 and 2.4μM, five times each,

while rinsing with PBS between concentrations.

The ratios, FI([s]i)/FI([s]j), between the measured FI, for different [s] and

fluorescein concentrations, were found to be in correlation to the ratios of FDA

substrate concentrations (fs]ι/[s]j) and free fluorescein concentrations ([F]i/[F]j),

(see Table 1).

The correlation between the substrate concentration (FDA concentration) and the

measured staining rates by intracellular fluorescein was established for cells.

First, each CC was loaded with unstained (BPS free of substrate) ceUs and stained with one chosen substrate concentrations (in order to avoid possible

influences of additive staining when sequential exposure is performed).

Second the sequential staining manipulation was examined. As can be seen in

the third and forth column of Table 1, there was good correlation between the

increasing staining rates (which means increasing rate of product formed) and

the increasing substrate concentrations in both cases.

Next, using the sequential staining manipulation [adding in sequence of different

concentrations of substrate and measuring the production rate of F in between

additions, by monitoring FI(t)] with cells, it was verified the theory described in

equation 7 specifically for the four cases that are detailed above and are

presented as simulations in Fig. 2.

First, cells were loaded on the CC and stained with FDA. Re-washing the cells

after every five or six scans with the same FDA concentration gave similar slopes

after every wash as can be seen for FDA at 1.5μM in Fig. 3a.

Rinsing (R) the cells with FDA and with PBS (no FDA, N=0, equation 7)

alternately gave similar slopes when FDA was present and almost zero slope

when FDA was absent, as shown in Fig 3b.

The level of FI at the beginning of the last rinse was higher than the level at the

end of the rinse with PBS though the slopes (velocities which are the magnitude

used for KMAPP determination) were identical. This difference is probably due to

technical reasons such as a slight change in the focus while manipulating FDA concentration or by laser beam geometrical instability etc. In Fig. 3c the cells

were rinsed with increasing FDA concentration of 0.6, 1.2, 2.4 and 3.6μM.

In Fig. 3d, the cells were rinsed with FDA at concentration of 0.6, 1.2, 2.4 μM and

in between with PBS without FDA. The PBS gave almost zero slopes (no

production of FI) while the increasing FI slopes were in good correlation with the

increasing FDA concentration. Generally, as can be seen from figures 2 and 3,

there was good correlation between the theoretical simulation and results of the

experiments.

Table 1

Table 1: Ratios of substrate concentrations (a); of fluorescein solutions FI (b); and

of intracellular fluorescein production rates of trapped cells: (c) parallely exposed

on different CC each to different FDA concentrations and (d) sequentially exposed

to different FDA concentrations on the same CC. Determination of Individual KMAPP and VMAX Values:

Determination of KMAPP and VMA_X was carried out by utihzing the reciprocal of

Eq. 8 (Lineweaver - Burk plot)

Thus, the use of two substrate concentrations should be, in principal, enough for

the extraction of KM and VMAX . Minimization of possible experimental errors,

while restricted by the linear range of time duration, Δt ≤ 10³ sec, led to the

choice of 4 FDA concentrations: 0.6, 1.2, 2.4 and 3.6 μM. Practically, trapped cells

on the cell carrier were sequentially exposed to the four FDA concentrations

staining solutions and scanned for determination of released fluorescein six

times per the same FDA concentration. A representative chart of a complete

measurement procedure made on 50 cells is shown in Fig. 4.

A plot of Eq. 8 for two cells out of the measured population of Fig. 4 is presented

in Fig. 5.

Example 2.

Utilization of Individual KMAPP Measurements

The Influence of Mitogenic Stimulation upon KMAPP and VMAX The activation of lymphocytes is a critical stage in most immune responses and

allows these cells to exert their specific functional capabilities. During activation,

the resting lymphocytes undergo complex changes resulting in cell differentiation

and proliferation. Lymphocytes activation is triggered by multiple interactions

that occur at the cell surface, which initiate intracellular biochemical events

within the cell that culminate in cellular response.

One of the experimental models used to study lymphocytes activation is lectins,

plant derived proteins (including phytohemagglutinin PHA), that bind

carbohydrate groups at the cell surface and stimulate relevant receptors involved

in physiologic lymphocyte activation. Many pharmacological agents mimic or

inhibit some of the intracellular events associated with T cell activation. An

example is described herein for individual KMAPP measurement following

lymphocyte activation.

The sequential FDA hydrolysis experiment was executed fohowing incubation of

cells with and without phytohemagglutinin PHA. The distribution of individual

KMAPP and VMAX values for both cases are presented in Fig. 6a and 6b,

respectively. The average KMAPP and VMAX were found to be 4.88 μM and 1.50 μM

and 695 (intensity/sec) and 652 (intensity/sec), indicating a decrease of 69% in

KMAPP and 6% in VMAX values for PHA compared to the control. Both distributions

indicated cell heterogeneity having a CV of about 70%. For comparison purposes, at the average level, the FC (Beckton-Dickinson

FACSCalibur) was used to determine KMAPP and VMAX value averages taken over

the cell population following the protocol suggested by Watson, JV and Dive, C,

Enzyme Kinetics. Methods Cell Biol (1994) 41: 469-508. Four means of

intracellular fluorescence intensities (IFI) were calculated from data accumulated

along four time gates of 25 second each and 30 seconds apart, from which Vo was

extracted. This process was sequentially performed on five different aliquots of

cells (50 μl, at a concentration o ^'6x10⁶ cells/ml) each exposed to different FDA

concentrations (0.3, 0.6, 1.2, 1.8 and 2.4 μM). Introducing these average Vo in

values Eq. 8 yielded population average KMAPP and VMAX of 2.16μM and 4.32μM

and 6.6 and 5.83 in cells incubated with and without PHA. It should be noted

that while KMAPP is an intrinsic value, VMAX depends on the optoelelctronic

arrangement under use. Thus, obviously, at the population level, measurements

carried out both on FC and average calculated from individual cell KMAPP

measurement data yield similar KMAPP values, indicating the validity of the

invented methodology.

Example 3.

Using basicaUy the same procedure, it is possible to determine the fohowing

enzymes activity in the single, individual cell:

1. Proteases and Peptidases

Peptidases and proteases play essential roles in protein activation, cell regulation

and signaling, as well as in the generation of amino acids for protein synthesis or

utilization in other metabohc pathways. Typical peptidase substrates are short

peptides conjugated to fluorophores (like 7-Amino-4-methylcoumarin (AMC) or Rhodamine 110). In the presence of the enzyme, the fluorogenic part is released,

and may be easily determined by fluorescence measurements. One example of

peptidase is the cystein protease- Caspase which play a pivotal role in

programmed cell death.

AMC- and RllO-labeled peptidase substrates, permit the detection of apoptosis

by assaying for increases in caspase-3 and caspase-3— like protease activities. The

activation of caspase-3 (CPP32/apopain), which has a substrate selectivity for the

amino acid sequence Asp-Glu-Val-Asp (DEVD) and cleaves a number of different

proteins, including poly(ADP-ribose) polymerase (PARP), DNA-dependent protein

kinase, protein kinase C and actin, is important for the initiation of apoptosis.

Both substrates can be used to continuously measure the activity of caspase-3.

2. Peroxidases

Reactive oxygen species, including singlet oxygen, superoxide, hydroxyl radical

and various peroxides (ROOR') and hydroperoxides (ROOH).are produced during

a number of physiological processes. Activated oxygen species react with a large

variety of easily oxidizable cellular components, including NADH, NADPH, dopa,

ascorbic acid, histidine, tryptophan, tyrosine, cysteine, glutathione, proteins and

nucleic acids. Reactive oxygen species can also oxidize cholesterol and

unsaturated fatty acids, causing membrane lipid peroxidation. The importance of

the nitric oxide radical enzyme producer and other reactive oxygen species as

biological messengers has been increasingly recognized during the last several

years. Assaying of oxidative activity in hve cells can be done by using Leuco

Dyes. Fluorescein, rhodamine and various other dyes can be chemically reduced to colorless, non-fluorescent leuco dyes. These "dihydro" derivatives are readily

oxidized back to the parent dye by some reactive oxygen species and thus can

serve as fluorogenic probes for detecting oxidative activity in cells.

Dihydroethidium, chcMorodihydrofluorescein (H2DCF) and dihydrorhodamine

123 react with intracellular hydrogen peroxide — a reaction mediated by

peroxidase, cytochrome C or Fe²⁺. The leuco dyes also serve as fluorogenic

substrates for peroxidase enzymes.

3. Glucose Oxidase

The enzyme glucose oxidase is widely used for glucose determination. Glucose

oxidase reacts with glucose to form gluconolactone and H2O2. The H2O2 is then

detected using fluorescent probe as described above.

4. Carbonic Anhydrase

Carbonic anhydrase catalyzes the reversible hydration of CO2 to carbonic acid.

Acetazolamide has been shown to bind carbonic anhydrases in a wide variety of

eukaryotic cells. Fluorescent-labeled derivative of acetazolamide is used for

studying carbonic anhydrase activity in live cells.

As was described hereinabove, a major embodiment of present invention involves

the measurement in individual cells of KMAPP and VMAX values of particular

cellular enzymes. This is a rather important assay relating to drug activity within a single intact cell.

In general, pre drug-treated cells are exposed to at least 2 different substrate

concentrations in order to determine the enzymatic KMA_PP and VMA_X values. The same cells are then exposed to the investigated drug (or any other biologically

active material, such as ,inducer, inhibitor,etc.), during a selected period of time.

Finally, the cells are again exposed either to the same 2 substrate concentrations

or another 2 or more substrate concentrations, and the KMAPP and VMAX values of

the drug-treated cells, is determined.

In the following, an example is given in order to demonstrate this principle.

Peripheral blood lymphocytes were loaded on a CC, and exposed to FDA, after

which individual FI(t) was measured. The same trapped cells, on the same CC

were then rinsed (R) twice with fresh buffer and incubated at 37°C in the

presence_of hydrogen peroxide (an apoptotic inducer). At the end of incubation,

the same cells were again exposed to the same FDA concentration and FI(t)

measurements were again performed.

Despite the fact that such an experimental procedure is self consistent (since it

has its own control on an individual cell basis, namely control measurements of

KMAPP and VMAX of ceUs, prior to their incubation with the drug), an additional

experiment was carried out as a second external control, but this time cells were

incubated without the drug.

FI(t) of two representative cells, measured prior to and after incubation with

(treated) and without (control) hydrogen peroxide (the drug) are shown in Fig. 7.

Since cells are in general heterogeneous, one would expect a distribution of FI(t)

rates (slopes) in the same experiment. This is why the initial slopes (Vo) of the two curves in Fig. 7 are not identical. Thus, in such an experimental procedure,

the determining parameter is the ratio between the initial and the final slopes-,

namely, the ratios between FI(t) slopes prior to and after incubation (with and

without drugs), as well as ratios of individual KMAPP and VMAX prior to and after

incubation.

Calculation of both slope ratios shown in Fig. 7 indicates that exposure of

lymphocytes to mild oxidative stress resulted in a lower rate of the second

staining reaction, in comparison to control. The ratio between the first and the

second reactions reflected the apoptotic activity of the inducer Moreover, it can

provide an idea regarding apoptotic resistance of specific individual cells.

Claims

1. A process for measuring enzymatic activity in an identified, isolated, intact,

single, viable cell, comprising the steps:

(a) placing each of the viable cells within individual identified locations on a

carrier of a cytometer having means to measure enzymatic activity of a single

viable cell placed in an identified location,

(b) exposing the identified isolated cell to a substrate of an enzyme to be

measured, and

(c) measuring the rate of product formed or released following every exposure of

the cell to same or different concentrations of the substrate.

2. A process according to claim 1, wherein the isolated cell is exposed to a

sequence of at least two different concentrations of the substrate and for each

exposure the rate of product formed or released, is measured.

3. A process according to claim 2 for measuring the kinetic of a particular

enzyme, wherein initial rate production (Vo-velocities) are measured from which

VMAX and KM are calculated.

4. A process according to claim 1, wherein activities of several different enzymes

are measured in the same isolated cell in a population..

5. A process according to claim 1, wherein activity of a particular enzyme is

measured before and after the treatment of said isolated cell with a biologicaUy

active material.

6. A process according to claim 5, wherein the biologically active material is a

drug.

7. A process according to claim 5, wherein the biologicaUy active material is an

inhibitor of any of the treated cell's functions.

8. A process according to claim 5, wherein the biologically active material

stimulates, induces or promotes a particular function or property of the treated

cell.

9. A process according to claim 5, wherein the production rates (Vo) are

measured and VMAX and KM are calculated before and after ceU treatments.

10. A process according to claim 1, wherein the substrate consists of a known

fluorescent substance that as a result of enzymatic activity is converted into a

measurable fluorescentic product.

11. A process according to claim 10, wherein the substrate is fluorescein- diacetate (FDA).

12. A process according to claim 1, wherein the measured activity is of an

intra-cellular enzyme.

13. A process according to claim 12, wherein the intra-cellular enzyme is selected

from the group comprising esterase, protease, peptidase, peroxidase, glucose

oxidase and carbonic anhydrase.

14. A process according to claim 1, wherein the measured activity is of an

extra-cellular enzyme.

15. A process according to claim 1 wherein the isolated single cell is a

lymphocyte.

16. A process according to claim 1, wherein the isolated single cell is a

lymphocyte, the enzyme is an esterase and the substrate is fluorescein-diacetate.

17. A process according to claim 1, wherein the substrate is color-less and the

product formed or released is colored.

18. A process according to claim 1, wherein the substrate is colored and the

product formed or released is colorless.